A Multiplex Bead-Based Immunoassay to Quantify Multiple Cytokine Targets

Take a multiwell plate with filters at the base.

Add a test sample containing different cytokines.

Add different sets of microspheres differentiated by size and internal fluorescence intensity.

Each set is conjugated to an antibody targeting a specific cytokine.

Incubate the plate in the dark under agitation.

The antibodies bind to their target cytokines, immobilizing them on the microspheres.

Apply a vacuum to remove the unbound cytokines. Microsphere-bound cytokines are retained due to the smaller pore size of the membrane.

Add a cocktail of biotin-conjugated detection antibodies that bind to the microsphere-bound cytokines.

Add a fluorescent reporter-conjugated streptavidin and incubate in the dark under agitation. Streptavidin binds to biotin, immobilizing the reporter on the microsphere complex.

Remove unbound molecules and resuspend the microspheres.

Using flow cytometry, identify the bound cytokines by determining the size and internal fluorescence of the microspheres.

To determine the amount of a specific cytokine, measure the reporter's fluorescence intensity.